Kits for Stabilization of cfDNA in Urine Samples

ABSTRACT

Disclosed here are kits comprising pre-packed stabilizing solutions for stabilizing combinations of biomarkers demonstrating sufficient accuracy and specificity for identifying kidney injuries. Such kits can be better adapted for sample collection at a subject&#39;s dwelling, thus easing the burdensome requirement of continuous monitoring for kidney injury.

CROSS-REFERENCE

The present application claims priority to International PatentApplication No. PCT/US21/63937, filed on Dec. 17, 2021, which claimspriority to U.S. Provisional Application Ser. No. 63/127,122, filed Dec.17, 2020, the contents of both of which are hereby incorporated byreference in their entireties.

BACKGROUND

Analyte measurement in physiological fluids, e.g., urine or bloodderived products, has important uses in a variety of applications,including clinical laboratory testing, home testing, etc., where theresults of such testing play a prominent role in the diagnosis andmanagement in a variety of disease conditions, including management ofchronic conditions such as chronic kidney disease (CKD) andtransplantation medicine.

According to the Centers for Disease Control and Prevention, kidneyfiltration begins to decrease by one percent every year after anindividual's 40^(th) birthday. But sometimes that process can acceleratewithout noticeable signs. Chronic kidney disease (CKD)—in which thekidneys filter fewer wastes from the blood, causing them to build up inthe body—can develop and proceed relatively symptom-free until yourkidneys are badly damaged.

There is a critical unmet need for improved non-invasive diagnosis forthe management and treatment of chronic conditions such as chronicconditions and organ transplantation.

SUMMARY

In some aspects, the disclosure provides a kit for the stabilization ofa urine sample from a subject comprising: a vacutainer cup for thecollection of the urine sample from the subject, the vacutainer cuphaving an inner protrusion functionally connected to a piercing hollowchannel; and at least one urine sample collection tube having a volumeof a pre-packaged solution or a pre-packaged powder for stabilizing atleast one analyte in the urine sample, whereby the at least one urinesample collection tube has a top configured to form a suction vacuumwhen pierced by the piercing hollow channel. In preferred embodimentsthe analyte is a cell-free nucleic acid, protein, or both. In otherembodiments, the analyte can be a cellular nucleic acid, e.g., mRNA, Inmost preferred embodiments the analyte is stable for at least 5 days atroom temperature, including temperatures up to 86° F. The pre-packagedstabilizers in the collection tube can be provided in solution form orin powder form. Alternatively, the pre-packed solution can be also besprayed onto the sides of the collection tube. These stabilizers arepreferably used for stabilizing the cell-free nucleic acid in the urinesample, including methylated cell-free DNA, and the total protein in theurine sample and generally comprise formaldehyde, a formaldehydequenching solution, a chelator and an agent that inhibits bacterialgrowth. In certain instances the kit comprises a second urine samplecollection tube, also comprising pre-packaged stabilizers. The secondurine collection sample generally comprises, a polyol, a proteincrowding stabilizer, and a chelator. In most preferred cases, thestabilizers in the second tube are specifically designed to stabilize atleast one additional biomarker, and the at least one additionalbiomarker is distinct from the cell-free DNA. In some cases, thecell-free DNA is not stable in the stabilizer used to pre-package thesecond stabilizer tube. The second urine sample collection tube maycomprise a volume of a second pre-packaged solution or an amount of apre-packaged powder for stabilizing at least one additional biomarker.In some cases, the second pre-packaged solution is for stabilizing aninflammation marker in the urine sample, such as CXCL10 or CXCL9. Inother cases, the second pre-packaged solution is for stabilizing anapoptotic marker in the urine sample, such as clusterin. In other cases,the second pre-packaged solution is for stabilizing a metabolite in theurine sample, such as creatinine, and one or more dimethyl arginines(ADMA/SDMA). In most preferred cases, the second stabilizing solution iseffective at stabilizing an inflammation marker, an apoptotic marker, ora metabolite.

Preferably, the kit further comprises an envelope, a box, or a bag forshipping one or more urine sample collection tube(s) after urinecollection via a courier service, all of which may bepre-addressed—optionally with postage pre-paid—for postage to a urineanalysis laboratory via the courier. In instances where the urinecollection tube is pre-packed with a solution, the volume of thepre-packaged solution may ranges from 0.5 milliliters to 4 milliliters,preferably providing for a 5-fold dilution of the urine sample in orderto achieve a desirable concentration ratio of stabilizer/urine.Specifically, the urine sample collection tube may be a 5 millilitercollection tube, a 10 milliliter collection tube, or a 12 millilitercollection tube all of which can be pre-packed with a solution or apowder that provides for a suitable final concentration of urine tostabilizer ratio. Alternatively, the pre-packed solution can be also besprayed onto the sides of the collection tube.

The vacutainer cup itself is specifically designed for the collection ofthe urine sample from the subject. For instance, in many cases, thepiercing hollow channel may not be suitable for a blood draw. Further,the stabilizer may not contain heparin or sodium fluoride (NaF), as itis not designed for a blood draw. Rather, the stabilizer solution maycomprise a nuclease inhibitor in a concentration sufficient to inhibitnucleases in the urine sample. Further, the stabilizer solution maycomprise a formaldehyde donor, a quencher, and a chelator in aconcentration sufficient to inhibit cell lysis and to inhibit nucleasesin the biofluid sample. Alternatively, the solution may comprise achelator, a polyol, and sodium azide in a concentration sufficient toinhibit cell lysis and to inhibit nucleases in the biofluid sample.

In many cases, the status of an organ of the subject is being monitoredwith a kit of the disclosure. Specifically, in preferred embodiments thesubject has, or is suspected of having organ injury. For instance, thesubject may use a kit of the disclosure to monitor the status of asurgical procedure, such as in cases where the subject received an organtransplant and is being monitored for potential rejection of theallograft. In such cases, the subject may receive a kit with two urinecollection tubes, where the first urine collection tube is configures tostabilize cell-free nucleic acid markers in the urine and the secondtube is configured to stabilize, for example, one or more of aninflammation marker, an apoptotic marker, or a metabolite in the urinesample. In other cases, the subject may have been ill, such as a subjectwho has recovered from Sars-CoV-2. In such instances, the subject mayreceive a kit with one tube for collection and monitoring of a cell-freeDNA marker that could reflect injury caused by the virus, such asinjuries caused by low oxygenation of the kidney. In preferred cases,the kit further comprises instructions for using the same. In mostpreferred cases, the instructions provide guidance for a subject thathas either received an organ transplant or is afflicted with chronickidney disease (CKD) on how to use the kit.

In some aspects, the disclosure provides a method for stabilizing aurine sample of a subject, the method comprising: providing, by thesubject, a urine sample in a vacutainer cup having an inner protrusionfunctionally connected to a piercing hollow channel; contacting, by thesubject, at least one urine sample collection tube having a volume of apre-packaged solution or a pre-packaged powder for stabilizing at leastone analyte in the urine sample with the inner protrusion of thevacutainer cup, whereby the at least one urine sample collection tubeforms a suction vacuum when piercing the piercing hollow channel,whereby the at least one urine sample collection tube suctions an amountof urine from the vacutainer cup to provide a collected urine sample;and remitting, by the subject, the collected urine sample to alaboratory for analysis.

Accordingly, disclosed herein are kits and methods for collecting aurine sample at a location, such as a subjects dwelling, and stabilizingsolutions that support a subsequent analysis complex multivariateanalysis of biomarkers from the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1 (FIG. 1) illustrates representative components of a kit of thedisclosure. Specifically, FIG. 1 illustrates two urine sample collectiontubes of different sizes pre-packed with suitable stabilizer solutions,a lid of a vacutainer cup having an inner protrusion with screw ridgesthat can functionally connect to the loose piercing hollow channel.

FIG. 2 (FIG. 2) illustrates representative components of a kit of thedisclosure. Specifically, FIG. 2 illustrates two urine sample collectiontubes of different sizes, an unassembled vacutainer cup having an innerprotrusion.

FIG. 3 (FIG. 3) illustrates representative dimensions of urine samplecollection tubes used with a kit of the disclosure.

FIG. 4 (FIG. 4) illustrates a subject opening a vacutainer, which can bedone prior to the subject providing a urine sample into the vacutainer.

FIG. 5 (FIG. 5) illustrates an inner protrusion of the vacutainerfunctionally connected to a piercing hollow channel. Each one of thecollection tubes illustrated in FIG. 3 can be pressed against thepiercing hollow channel for urine sample collection.

FIG. 6 (FIG. 6) illustrates an assembled vacutainer of the disclosure.

FIG. 7 (FIG. 7) illustrates suction of the urine sample by thevacutainer and inversion of the sample for mixing. FIG. 7A illustratesthe insertion of a urine collection tube into a vacutainer. FIG. 7Billustrates the inversion of the urine collection tube back-and-forthrepeatedly for a sufficient number of times.

FIG. 8 (FIG. 8) illustrates the labeling of individual tubes andinsertion of each tube into a sleeve.

FIG. 9 (FIG. 9) illustrates the shipping process of samples with anexemplary kit of the disclosure.

FIG. 10 (FIG. 10) illustrates cfDNA/m-cfDNA Stability at RT (n=30).

FIG. 11 (FIG. 11) illustrates Clusterin Stability at RT (n=24).

FIG. 12 (FIG. 12) illustrates Creatinine Stability at RT (n=29)

FIG. 13 (FIG. 13) illustrates CXCL10 Stability at RT (n=26).

FIG. 14 (FIG. 14) illustrates Total Protein Stability at RT (n=29).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

DETAILED DESCRIPTIONS

Urine, a biofluid produced by the kidneys, can be a source ofinformative biomarkers for kidney health, injury, and disease. Thekidneys, collectively known as the renal system, perform the essentialfunction of removing waste products from the blood and regulating thewater fluid levels. They are essential in the urinary system, but alsoserve homeostatic functions such as the regulation of electrolytes,maintenance of acid-base balance, and regulation of blood pressure. Theyserve the body as a natural filter of the blood, and remove wastes whichare diverted to the urinary bladder. In producing urine, the kidneysexcrete wastes such as urea and ammonium, and they are also responsiblefor the reabsorption of water, glucose, and amino acids. If the rightbiomarker, or combination of biomarkers, is (are) identified, a urinesample can provide a suitable, non-invasive source of material for theevaluation of a solid allograft status. Urine can contain sufficientbiomarkers to inform the status of kidney allografts with highsensitivity and accuracy, and it may be able to inform the status ofother allografts as well.

Sarwal and colleagues investigated uses of various samples, includingurine, as non-invasive sources of other informative biomarkers for themonitoring of different types of solid organ transplants (See, e.g.,Sarwal WO2014/145232). Sarwal recognized that Alu elements are the mostabundant transposable elements in the human genome, containing over onemillion copies dispersed throughout the human genome. Recognizing theabundance of ALU repeats, Sarwal created a ratio of ALU repeats in aurine sample of a transplant patient over the number of ALU repeats in aurine sample from a normal population. The ratio could be used as aproxy of injury, however, on its own it was not sufficientlyinformative.

Subsequent studies explored potential combination of biomarkers asproxy's for allograft injury. For instant, QSant™ utilizes a compositescore of various biomarkers of distinct biochemical characteristics,i.e., proteins, metabolites, and nucleic acids. (See Yang, Sarwal, etal., A urine score for noninvasive accurate diagnosis and prediction ofkidney transplant rejection. Science Translational Medicine, 18 Mar.2020, Vol. 12, Issue 535; see also WO20180/35340). Yang et al.demonstrated that a urinary composite score of six biomarkers—aninflammation biomarker (CXCL-10, also known as IP-10); an apoptosisbiomarker (e.g., clusterin); a cfDNA biomarkers; a DNA methylationbiomarker; a creatinine biomarker; and total protein—enables diagnosisof Acute Rejection (AR), with a receiver-operator characteristic curvearea under the curve of 0.99 and an accuracy of 96%. Notably, QSant™predicts acute rejection before a rise in a stand-alone serum creatininetest, enabling earlier detection of rejection than currently possible bycurrent standard of care tests. Sarwal considered potential ways ofstabilizing the samples, but it failed to conjure a combination ofreagents that could stabilize a urine sample for any longer than 72hours. See, e.g., Sarwal WO20180/35340). This is important becauseanalyte instability in urine precludes the recipients of an allograftfrom providing a urine void while in the comfort of their own homes thatis sufficiently stable, for example, to be used in the analysisdescribed by Yang, Sarwal, et all. Id.

The traditional method of midstream urine collection is for thehealthcare professional to give the patient some sort of pot in which tocollect their urine; this could be a universal container, a small tub,or a cup which the collecting physician may or may not store indifferent containers. In the laboratory, the specimen is typicallydecanted again, into a secondary primary tube. One specimen cantherefore be decanted and handled two or three times before it reachesthe laboratory, and there is no guarantee it has not been storedproperly or under conditions that preserve the urine sample for furtheranalysis. Further, it is extremely difficult to control the amount ofsample being collected by a patient in a container. Further, patientsare not positioned to collect precise amounts of samples and combinesuch precise amounts with any other chemical reagent.

The present disclosure provides kits for the stabilization of aplurality of markers in a urine sample from a subject that can stabilizethe biomarkers in the urine sample for a suitable period-of-time suchthat the sample can be collected at a subject's home and analyzed dayslater at a laboratory. Such kits are created, designed, and configuredto collect defined amounts of urine samples in containers such thatthese amounts are mixed with preservatives pre-packaged in the containerin suitable ratios for preserving the urine sample. Preferably, suchkits can stabilize a urine sample for at least 5 days at roomtemperature. Preferably, such kits can stabilize a urine sample in arange of temperatures, including temperatures up to 86° F., to supportan analysis of a sample that has been exposed to varying temperatureswhile being routed to a laboratory.

In some cases, provided herein are vacutainer kits for both collectingand stabilizing a sample for at least 5 days at room temperature andabove. In preferred embodiments, a subject receives in the mail, by apostal courier, a urine testing kit comprising a vacutainer and one ormore collection tubes (FIGS. 1-3) pre-packaged with liquid forms, powderforms, or gel forms of a stabilizing solution. Alternatively, thepre-packed solution can be also be sprayed onto the sides of thecollection tube. In most preferred embodiments, the vacutainer cuphaving a screw top lid (FIG. 4) having an inner protrusion functionallyconnected to a piercing hollow channel (FIG. 5). Once the urine specimenis introduced into the vacutainer, the vacutainer's lid is closed thusforming a seal (FIG. 6). Each one of the individual urine samplecollection tubes is pressed against the piercing channel of thevacutainer which releases an amount of urine inside the collectiontubes. The collection tubes may be “inverted 5-10 times” until the urinesample is mixed with the pre-packaged stabilizer solution inside thecollection tube.

Stabilizing Urine Biomarkers in Collection Tubes of the Disclosed Kits

cfDNA and Total Protein Sample Collection Tubes

In one aspect, the instant disclosure provides a kit and methods forcollecting a urine sample comprising a cell-free DNA (cfDNA) and keepingit stable. Cell-free DNA naturally occurs in biofluids such as blood andhas been largely attributed to apoptotic and necrotic processes. Whilethe presence of cfDNA in blood was discovered in 1948, its implicationsin clinical medicine were not realized for more than two decades. Muchless is known about the abundance and the utility of cfDNA from urinesamples.

Since that time, a cumulative body of research has identified cfDNA andm-cfDNA as both a prognostic and diagnostic indicator for multiplepathogenic conditions; i.e., allograft rejection, kidney injury, andvarious cancers, See, e.g., Sarwal WO20180/35340). As such, accuratedetection of cfDNA and m-cfDNA in human biological specimens may providea non-invasive avenue that allows assessment, screening, and diseaseclassification and monitoring, if the outstanding challenges of keepingthe sample stable are successfully overcome. The detection of cfDNA,however, is particularly challenging for the following reasons: (1)sample processing after collection can induce cell lysis, which oftenleads to aberrant increases in the amount of circulating cfDNA, and (2)the relatively low level of cfDNA underscores the potential risks ofgenerating false negative results due to the loss of scarce target cfDNAsequences-due to sample instability or inappropriate sample processing.

Due to the low abundance of the cfDNA biomarkers in any sample, andespecially due to its particularly low abundance in urine samples, it isrecommended that genomic DNA (gDNA) background levels be minimized toprovide accurate measurements of cfDNA levels (the average circulatingconcentration of cfDNA for a healthy individual is 30 ng/ml, the cfDNAis generally double stranded, and approximately 0.18-21 kilobases insize (See Wagner, J. “Free DNA new potential analyte in clinicallaboratory diagnostics?” Biochem Med (Zagreb) 22(1): 24-38)). It isfurther beneficial that the structural integrity of the cfDNA bemaintained due to the minimal amounts available for analysis. It istherefore necessary to address several pre-analytical issues that ariseduring the time between urine collection and subsequent DNA isolation.These issues include the ability to combine suitable amounts of urinewith suitable ratios of preservatives, delays in urine processing, urinestorage temperature, and agitation of the sample during transport andshipment of urine. Such conditions may alter urinary DNA levels bycausing gDNA release from lysed bladder and uroepithelial cells andobfuscate true cfDNA. As a result, it is important to consider the typeof urine collection device and post-collection conditions while workingwith cfDNA samples.

The disclosure provided herein, provides strategies for combiningsuitable amounts of urine with an upper limit of volume defined by thecapacity of the container which overcomes challenges in having a subjectprovide a suitable amount of urine at home and combine it with asuitable amount of preservative (See FIGS. 1-8). In many aspects, thekits and vacutainer kits provided herein require a minimum amount ofpreservative to maintain the biomarker in a state that is suitable foranalysis. In many instances, an excess amount of preservative asprovided in a vacutainer of the disclosure does not affect the abilityof a biomarker to be further analyzed, but a minimum amount ofpreservative is required. Usefully, the format of the kit, namelyvacutainers pre-packed with a suitable concentration of a preservativefor a set of biomarkers, allow a subject to collect the urine sample athome, without the need of either a venipuncture or more challengingmanipulation of a suitable manipulation of urine to preservative ratiosin open containers by the subjects.

In preferred instances, the collection tubes that stabilize the cfDNAalso stabilize an amount of the total protein in the urine sample. Themeasurement of urine total protein is central to the diagnosis andmanagement of subjects with kidney disease/injury. For instance,proteinuria is a strong predictor of adverse cardiovascular and kidneyevents, and an accurate assessment of proteinuria is important for theevaluation and management of CKD. Proteinuria has been associated withtransplant loss and mortality in kidney transplant recipients. Both spotsamples (albumin-creatinine ratio (ACR) and protein-creatinine ratio(PCR) and 24-hour collections (albumin excretion rate (AER) and proteinexcretion rate (PER)) have been used to quantify protein excretion, butwhich measurement is a better predictor of outcomes in kidneytransplantation remains uncertain. The present disclosure provides a kitand methods for collecting urine samples and shipping them to alaboratory that stabilizes both a cfDNA and an amount of the totalprotein in a sample for at least 5 days at room temperature.

For blood and serum samples, the art discloses the addition ofanticoagulants such as heparin, sodium fluoride (NaF), and citrate aregenerally added to collection tubes to prevent clotting of whole bloodcells, which is thought to reduce DNA release from the leukocyte cellpopulation. Also, the optimization of centrifugation conditions isrequired to prevent lysis but adequately separate intact cells fromcell-free plasma. Because healthy urine samples generally have scantcellularity the disclosure provides better and distinct stabilizingsolutions that more effectively preserve the analyte, as opposed tomerely preserving cell death. In some aspects, the disclosure provides akit that has a urine collection tube pre-packaged with a formaldehydedonor. Typical formaldehyde donors used in urine collection tubes of thedisclosure include DMDM hydantoin (DMDMH), imadazolidinyl urea,diazlidinyl urea, sodium hydroxyl and methyl glycinate. In manyinstances, an excess amount of formaldehyde as provided in a vacutainerof the disclosure does not affect the ability of the particularbiomarker to be further analyzed, but a minimum amount of preservativeis required. Pre-packaging in the vacutainers of the disclosure providesa suitable amount of preservative for combining with a volume of urinethat is in the range of 1 mL to 20 mLs; in the range of 1 mL to 15 mLs;in the range of 1 mL to 10 mLs; in the range of 1 mL to 5 mLs; oranother suitable amounts within those ranges.

Alternatively the formaldehyde donor could be added to the collectiontube in powder format. In these instances, the concentration of thepowder in the rube would be calculated accordingly to provide a dilutionratio of about 1:5. The urine sample collection tube can be a 5milliliter collection tube, a 10 milliliter collection tube, a 12milliliter collection tube, or another suitable size and theconcentrated amount of formaldehyde can be adjusted accordingly toprovide a dilution ratio of about 1:5.

Addition of a formaldehyde donor may prevent subsequent amplification ofsome cfDNA genes, notheless in preferred instances the subsequentdetection of cfDNA in the urine samples does not require amplification.In such instances, presence of cfDNA in a sample can be inferred fromthe presence of an Alu repeat—or another suitable region—in the cfDNAwithout amplification. Alu elements belong to a primate specific classof retroelements termed SINEs (short interspersed elements). There areover one million Alu elements interspersed throughout the human genome,and it is estimated that about 10.7% of the human genome consists of Alusequences. However, less than 0.5% are polymorphic (i.e., occurring inmore than one form or morph). The typical structure of an Alu element is5′-Part A—ASTACA6-Part B—PolyA Tail-3′, where Part A and Part B (alsoknown as “left arm” and “right arm”) are similar nucleotide sequences.Alu repeats are similarly abundantly present in cfDNA. Thus, detectionof Alu sequences from short cfDNA fragments can be used as a biomarkerfor detection of cfDNA, even if the sample has been stabilized with asolution that otherwise, for example, includes a formaldehyde donor.

A urine collection tube of the disclosure may be pre-packed withanywhere from: 25 g/L

-   -   800 g/L of a formaldehyde donor, 50 g/L-800 g/L of a        formaldehyde donor, 75 g/L-800 g/L of a formaldehyde donor, 100        g/L-800 g/L of a formaldehyde donor, 125 g/L-800 g/L of a        formaldehyde donor, 150 g/L-800 g/L of a formaldehyde donor, 175        g/L-800 g/L of a formaldehyde donor, 200 g/L-800 g/L of a        formaldehyde donor, 225 g/L-800 g/L of a formaldehyde donor, 250        g/L-800 g/L of a formaldehyde donor, 275 g/L-800 g/L of a        formaldehyde donor, 300 g/L-800 g/L of a formaldehyde donor, 325        g/L-800 g/L of a formaldehyde donor, 350 g/L-800 g/L of a        formaldehyde donor, 375 g/L-800 g/L of a formaldehyde donor, 400        g/L-800 g/L of a formaldehyde donor, 425 g/L-800 g/L of a        formaldehyde donor, 450 g/L-800 g/L of a formaldehyde donor, 475        g/L-800 g/L of a formaldehyde donor, 500 g/L-800 g/L of a        formaldehyde donor, 525 g/L-800 g/L of a formaldehyde donor, 550        g/L-800 g/L of a formaldehyde donor, 575 g/L-800 g/L of a        formaldehyde donor, 600 g/L-800 g/L of a formaldehyde donor, 625        g/L-800 g/L of a formaldehyde donor, 650 g/L-800 g/L of a        formaldehyde donor, 675 g/L-800 g/L of a formaldehyde donor, 700        g/L-800 g/L of a formaldehyde donor, 725 g/L-800 g/L of a        formaldehyde donor, 750 g/L-800 g/L of a formaldehyde donor, 775        g/L-800 g/L of a formaldehyde donor, 50 g/L-700 g/L of a        formaldehyde donor, 75 g/L-700 g/L of a formaldehyde donor, 100        g/L-700 g/L of a formaldehyde donor, 125 g/L-700 g/L of a        formaldehyde donor, 150 g/L-700 g/L of a formaldehyde donor, 175        g/L-700 g/L of a formaldehyde donor, 200 g/L-700 g/L of a        formaldehyde donor, 225 g/L-700 g/L of a formaldehyde donor, 250        g/L-700 g/L of a formaldehyde donor, 275 g/L-700 g/L of a        formaldehyde donor, 300 g/L-700 g/L of a formaldehyde donor, 325        g/L-700 g/L of a formaldehyde donor, 350 g/L-700 g/L of a        formaldehyde donor, 375 g/L-700 g/L of a formaldehyde donor, 400        g/L-700 g/L of a formaldehyde donor, 425 g/L-700 g/L of a        formaldehyde donor, 450 g/L-700 g/L of a formaldehyde donor, 475        g/L-700 g/L of a formaldehyde donor, 500 g/L-700 g/L of a        formaldehyde donor, 525 g/L-700 g/L of a formaldehyde donor, 550        g/L-700 g/L of a formaldehyde donor, 575 g/L-700 g/L of a        formaldehyde donor, 600 g/L-700 g/L of a formaldehyde donor, 625        g/L-700 g/L of a formaldehyde donor, 650 g/L-700 g/L of a        formaldehyde donor, 675 g/L-700 g/L of a formaldehyde donor, 50        g/L-600 g/L of a formaldehyde donor, 75 g/L-600 g/L of a        formaldehyde donor, 100 g/L-600 g/L of a formaldehyde donor, 125        g/L-600 g/L of a formaldehyde donor, 150 g/L-600 g/L of a        formaldehyde donor, 175 g/L-600 g/L of a formaldehyde donor, 200        g/L-600 g/L of a formaldehyde donor, 225 g/L-600 g/L of a        formaldehyde donor, 250 g/L-600 g/L of a formaldehyde donor, 275        g/L-600 g/L of a formaldehyde donor, 300 g/L-600 g/L of a        formaldehyde donor, 325 g/L-600 g/L of a formaldehyde donor, 350        g/L-600 g/L of a formaldehyde donor, 375 g/L-600 g/L of a        formaldehyde donor, 400 g/L-600 g/L of a formaldehyde donor, 425        g/L-600 g/L of a formaldehyde donor, 450 g/L-600 g/L of a        formaldehyde donor, 475 g/L-600 g/L of a formaldehyde donor, 500        g/L-600 g/L of a formaldehyde donor, 525 g/L-600 g/L of a        formaldehyde donor, 550 g/L-600 g/L of a formaldehyde donor, 575        g/L-600 g/L of a formaldehyde donor, 50 g/L-500 g/L of a        formaldehyde donor, 75 g/L-500 g/L of a formaldehyde donor, 100        g/L-500 g/L of a formaldehyde donor, 125 g/L-500 g/L of a        formaldehyde donor, 150 g/L-500 g/L of a formaldehyde donor, 175        g/L-500 g/L of a formaldehyde donor, 200 g/L-500 g/L of a        formaldehyde donor, 225 g/L-500 g/L of a formaldehyde donor, 250        g/L-500 g/L of a formaldehyde donor, 275 g/L-500 g/L of a        formaldehyde donor, 300 g/L-500 g/L of a formaldehyde donor, 325        g/L-500 g/L of a formaldehyde donor, 350 g/L-500 g/L of a        formaldehyde donor, 375 g/L-500 g/L of a formaldehyde donor, 400        g/L-500 g/L of a formaldehyde donor, 425 g/L-500 g/L of a        formaldehyde donor, 450 g/L-500 g/L of a formaldehyde donor, 475        g/L-500 g/L of a formaldehyde donor, 50 g/L-400 g/L of a        formaldehyde donor, 75 g/L-400 g/L of a formaldehyde donor, 100        g/L-400 g/L of a formaldehyde donor, 125 g/L-400 g/L of a        formaldehyde donor, 150 g/L-400 g/L of a formaldehyde donor, 175        g/L-400 g/L of a formaldehyde donor, 200 g/L-400 g/L of a        formaldehyde donor, 225 g/L-400 g/L of a formaldehyde donor, 250        g/L-400 g/L of a formaldehyde donor, 275 g/L-400 g/L of a        formaldehyde donor, 300 g/L-400 g/L of a formaldehyde donor, 325        g/L-400 g/L of a formaldehyde donor, 350 g/L-400 g/L of a        formaldehyde donor, 375 g/L-400 g/L of a formaldehyde donor, 50        g/L-300 g/L of a formaldehyde donor, 75 g/L-300 g/L of a        formaldehyde donor, 100 g/L-300 g/L of a formaldehyde donor, 125        g/L-300 g/L of a formaldehyde donor, 150 g/L-300 g/L of a        formaldehyde donor, 175 g/L-300 g/L of a formaldehyde donor, 200        g/L-300 g/L of a formaldehyde donor, 225 g/L-300 g/L of a        formaldehyde donor, 250 g/L-300 g/L of a formaldehyde donor. In        preferred instances, each collection tube in the kit is        pre-packed with wherein the volume of the pre-packaged solution        ranges from 0.5 milliliters to 4 milliliters. Such amounts        provide for a concentrated amount of formaldehyde that is        generally diluted about 5-fold by the addition of the urine        sample.

The urine collection sample of the disclosure may also be pre-packedwith a quenching agent to minimize protein-protein crosslinking that mayoccur due to formaldehyde addition. The quenching solution may be anyagent known to quench excess formaldehyde including, but not limited toglycine. In many instances, an excess amount of a quenching solution asprovided in a vacutainer of the disclosure does not affect the abilityof the particular biomarker to be further analyzed—it is largely used toquench the excess of formaldehyde in a sample—but a minimum amount ofpreservative is required. Pre-packaging in the vacutainers of thedisclosure provides a suitable amount of a quenching agent for combiningwith a volume of urine that is in the range of 1 mL to 20 mLs; in therange of 1 mL to 15 mLs; in the range of 1 mL to 10 mLs; in the range of1 mL to 5 mLs; or another suitable amounts within those ranges. A urinecollection tube of the disclosure may be pre-packed with anywhere from:2.5 g/L-80 g/L of a quenching agent, 5 g/L-80 g/L of a quenching agent,7.5 g/L-80 g/L of a quenching agent, 10 g/L-80 g/L of a quenching agent,12.5 g/L-80 g/L of a quenching agent, 15 g/L-80 g/L of a quenchingagent, 1.75 g/L-80 g/L of a quenching agent, 2 g/L-80 g/L of a quenchingagent, 2.25 g/L-80 g/L of a quenching agent, 2.5 g/L-80 g/L of aquenching agent, 2.75 g/L-80 g/L of a quenching agent, 3 g/L-80 g/L of aquenching agent, 3.25 g/L-80 g/L of a quenching agent, 3.5 g/L-80 g/L ofa quenching agent, 3.75 g/L-80 g/L of a quenching agent, 4 g/L-80 g/L ofa quenching agent, 4.25 g/L-80 g/L of a quenching agent, 4.5 g/L-80 g/Lof a quenching agent, 4.75 g/L-80 g/L of a quenching agent, 5 g/L-80 g/Lof a quenching agent, 5.25 g/L-80 g/L of a quenching agent, 5.5 g/L-80g/L of a quenching agent, 5.75 g/L-80 g/L of a quenching agent, 6 g/L-80g/L of a quenching agent, 6.25 g/L-80 g/L of a quenching agent, 6.5g/L-80 g/L of a quenching agent, 6.75 g/L-80 g/L of a quenching agent, 7g/L-80 g/L of a quenching agent, 7.25 g/L-80 g/L of a quenching agent,7.5 g/L-80 g/L of a quenching agent, 8 g/L-80 g/L of a quenching agent,8.25 g/L-80 g/L of a quenching agent, 8.5 g/L-80 g/L of a quenchingagent, 8.75 g/L-80 g/L of a quenching agent, 9 g/L-80 g/L of a quenchingagent, 9.25 g/L-80 g/L of a quenching agent, 9.5 g/L-80 g/L of aquenching agent, 9.75 g/L-80 g/L of a quenching agent, 10 g/L-80 g/L ofa quenching agent, 10.25 g/L-80 g/L of a quenching agent, 10.5 g/L-80g/L of a quenching agent, 10.75 g/L-80 g/L of a quenching agent, 11g/L-80 g/L of a quenching agent, 11.25 g/L-80 g/L of a quenching agent,11.5 g/L-80 g/L of a quenching agent, 11.75 g/L-80 g/L of a quenchingagent, 12 g/L-80 g/L of a quenching agent, 12.25 g/L-80 g/L of aquenching agent, 12.5 g/L-80 g/L of a quenching agent, 12.75 g/L-80 g/Lof a quenching agent, 13 g/L-80 g/L of a quenching agent, 13.25 g/L-80g/L of a quenching agent, 13.5 g/L-80 g/L of a quenching agent, 13.75g/L-80 g/L of a quenching agent, 14 g/L-80 g/L of a quenching agent,14.25 g/L-80 g/L of a quenching agent, 14.5 g/L-80 g/L of a quenchingagent, 14.75 g/L-80 g/L of a quenching agent, 15 g/L-80 g/L of aquenching agent, 2.5 g/L-70 g/L of a quenching agent, 5 g/L-70 g/L of aquenching agent, 7.5 g/L-70 g/L of a quenching agent, 10 g/L-70 g/L of aquenching agent, 12.5 g/L-70 g/L of a quenching agent, 15 g/L-70 g/L ofa quenching agent, 1.75 g/L-70 g/L of a quenching agent, 2 g/L-70 g/L ofa quenching agent, 2.25 g/L-70 g/L of a quenching agent, 2.5 g/L-70 g/Lof a quenching agent, 2.75 g/L-70 g/L of a quenching agent, 3 g/L-70 g/Lof a quenching agent, 3.25 g/L-70 g/L of a quenching agent, 3.5 g/L-70g/L of a quenching agent, 3.75 g/L-70 g/L of a quenching agent, 4 g/L-70g/L of a quenching agent, 4.25 g/L-70 g/L of a quenching agent, 4.5g/L-70 g/L of a quenching agent, 4.75 g/L-70 g/L of a quenching agent, 5g/L-70 g/L of a quenching agent, 5.25 g/L-70 g/L of a quenching agent,5.5 g/L-70 g/L of a quenching agent, 5.75 g/L-70 g/L of a quenchingagent, 6 g/L-70 g/L of a quenching agent, 6.25 g/L-70 g/L of a quenchingagent, 6.5 g/L-70 g/L of a quenching agent, 6.75 g/L-70 g/L of aquenching agent, 7 g/L-70 g/L of a quenching agent, 7.25 g/L-70 g/L of aquenching agent, 7.5 g/L-70 g/L of a quenching agent, 8 g/L-70 g/L of aquenching agent, 8.25 g/L-70 g/L of a quenching agent, 8.5 g/L-70 g/L ofa quenching agent, 8.75 g/L-70 g/L of a quenching agent, 9 g/L-70 g/L ofa quenching agent, 9.25 g/L-70 g/L of a quenching agent, 9.5 g/L-70 g/Lof a quenching agent, 9.75 g/L-70 g/L of a quenching agent, 10 g/L-70g/L of a quenching agent, 10.25 g/L-70 g/L of a quenching agent, 10.5g/L-70 g/L of a quenching agent, 10.75 g/L-70 g/L of a quenching agent,11 g/L-70 g/L of a quenching agent, 11.25 g/L-70 g/L of a quenchingagent, 11.5 g/L-70 g/L of a quenching agent, 11.75 g/L-70 g/L of aquenching agent, 12 g/L-70 g/L of a quenching agent, 12.25 g/L-70 g/L ofa quenching agent, 12.5 g/L-70 g/L of a quenching agent, 12.75 g/L-70g/L of a quenching agent, 13 g/L-70 g/L of a quenching agent, 13.25g/L-70 g/L of a quenching agent, 13.5 g/L-70 g/L of a quenching agent,13.75 g/L-70 g/L of a quenching agent, 14 g/L-70 g/L of a quenchingagent, 14.25 g/L-70 g/L of a quenching agent, 14.5 g/L-70 g/L of aquenching agent, 14.75 g/L-70 g/L of a quenching agent, 15 g/L-70 g/L ofa quenching agent, 2.5 g/L-60 g/L of a quenching agent, 5 g/L-60 g/L ofa quenching agent, 7.5 g/L-60 g/L of a quenching agent, 10 g/L-60 g/L ofa quenching agent, 12.5 g/L-60 g/L of a quenching agent, 15 g/L-60 g/Lof a quenching agent, 1.75 g/L-60 g/L of a quenching agent, 2 g/L-60 g/Lof a quenching agent, 2.25 g/L-60 g/L of a quenching agent, 2.5 g/L-60g/L of a quenching agent, 2.75 g/L-60 g/L of a quenching agent, 3 g/L-60g/L of a quenching agent, 3.25 g/L-60 g/L of a quenching agent, 3.5g/L-60 g/L of a quenching agent, 3.75 g/L-60 g/L of a quenching agent, 4g/L-60 g/L of a quenching agent, 4.25 g/L-60 g/L of a quenching agent,4.5 g/L-60 g/L of a quenching agent, 4.75 g/L-60 g/L of a quenchingagent, 5 g/L-60 g/L of a quenching agent, 5.25 g/L-60 g/L of a quenchingagent, 5.5 g/L-60 g/L of a quenching agent, 5.75 g/L-60 g/L of aquenching agent, 6 g/L-60 g/L of a quenching agent, 6.25 g/L-60 g/L of aquenching agent, 6.5 g/L-60 g/L of a quenching agent, 6.75 g/L-60 g/L ofa quenching agent, 7 g/L-60 g/L of a quenching agent, 7.25 g/L-60 g/L ofa quenching agent, 7.5 g/L-60 g/L of a quenching agent, 8 g/L-60 g/L ofa quenching agent, 8.25 g/L-60 g/L of a quenching agent, 8.5 g/L-60 g/Lof a quenching agent, 8.75 g/L-60 g/L of a quenching agent, 9 g/L-60 g/Lof a quenching agent, 9.25 g/L-60 g/L of a quenching agent, 9.5 g/L-60g/L of a quenching agent, 9.75 g/L-60 g/L of a quenching agent, 10g/L-60 g/L of a quenching agent, 10.25 g/L-60 g/L of a quenching agent,10.5 g/L-60 g/L of a quenching agent, 10.75 g/L-60 g/L of a quenchingagent, 11 g/L-60 g/L of a quenching agent, 11.25 g/L-60 g/L of aquenching agent, 11.5 g/L-60 g/L of a quenching agent, 11.75 g/L-60 g/Lof a quenching agent, 12 g/L-60 g/L of a quenching agent, 12.25 g/L-60g/L of a quenching agent, 12.5 g/L-60 g/L of a quenching agent, 12.75g/L-60 g/L of a quenching agent, 13 g/L-60 g/L of a quenching agent,13.25 g/L-60 g/L of a quenching agent, 13.5 g/L-60 g/L of a quenchingagent, 13.75 g/L-60 g/L of a quenching agent, 14 g/L-60 g/L of aquenching agent, 14.25 g/L-60 g/L of a quenching agent, 14.5 g/L-60 g/Lof a quenching agent, 14.75 g/L-60 g/L of a quenching agent, 15 g/L-60g/L of a quenching agent, 2.5 g/L-50 g/L of a quenching agent, 5 g/L-50g/L of a quenching agent, 7.5 g/L-50 g/L of a quenching agent, 10 g/L-50g/L of a quenching agent, 12.5 g/L-50 g/L of a quenching agent, 15g/L-50 g/L of a quenching agent, 1.75 g/L-50 g/L of a quenching agent, 2g/L-50 g/L of a quenching agent, 2.25 g/L-50 g/L of a quenching agent,2.5 g/L-50 g/L of a quenching agent, 2.75 g/L-50 g/L of a quenchingagent, 3 g/L-50 g/L of a quenching agent, 3.25 g/L-50 g/L of a quenchingagent, 3.5 g/L-50 g/L of a quenching agent, 3.75 g/L-50 g/L of aquenching agent, 4 g/L-50 g/L of a quenching agent, 4.25 g/L-50 g/L of aquenching agent, 4.5 g/L-50 g/L of a quenching agent, 4.75 g/L-50 g/L ofa quenching agent, 5 g/L-50 g/L of a quenching agent, 5.25 g/L-50 g/L ofa quenching agent, 5.5 g/L-50 g/L of a quenching agent, 5.75 g/L-50 g/Lof a quenching agent, 6 g/L-50 g/L of a quenching agent, 6.25 g/L-50 g/Lof a quenching agent, 6.5 g/L-50 g/L of a quenching agent, 6.75 g/L-50g/L of a quenching agent, 7 g/L-50 g/L of a quenching agent, 7.25 g/L-50g/L of a quenching agent, 7.5 g/L-50 g/L of a quenching agent, 8 g/L-50g/L of a quenching agent, 8.25 g/L-50 g/L of a quenching agent, 8.5g/L-50 g/L of a quenching agent, 8.75 g/L-50 g/L of a quenching agent, 9g/L-50 g/L of a quenching agent, 9.25 g/L-50 g/L of a quenching agent,9.5 g/L-50 g/L of a quenching agent, 9.75 g/L-50 g/L of a quenchingagent, 10 g/L-50 g/L of a quenching agent, 10.25 g/L-50 g/L of aquenching agent, 10.5 g/L-50 g/L of a quenching agent, 10.75 g/L-50 g/Lof a quenching agent, 11 g/L-50 g/L of a quenching agent, 11.25 g/L-50g/L of a quenching agent, 11.5 g/L-50 g/L of a quenching agent, 11.75g/L-50 g/L of a quenching agent, 12 g/L-50 g/L of a quenching agent,12.25 g/L-50 g/L of a quenching agent, 12.5 g/L-50 g/L of a quenchingagent, 12.75 g/L-50 g/L of a quenching agent, 13 g/L-50 g/L of aquenching agent, 13.25 g/L-50 g/L of a quenching agent, 13.5 g/L-50 g/Lof a quenching agent, 13.75 g/L-50 g/L of a quenching agent, 14 g/L-50g/L of a quenching agent, 14.25 g/L-50 g/L of a quenching agent, 14.5g/L-50 g/L of a quenching agent, 14.75 g/L-50 g/L of a quenching agent,15 g/L-50 g/L of a quenching agent,

2.5 g/L-40 g/L of a quenching agent, 5 g/L-40 g/L of a quenching agent,7.5 g/L-40 g/L of a quenching agent, 10 g/L-40 g/L of a quenching agent,12.5 g/L-40 g/L of a quenching agent, 15 g/L-40 g/L of a quenchingagent, 1.75 g/L-40 g/L of a quenching agent, 2 g/L-40 g/L of a quenchingagent, 2.25 g/L-40 g/L of a quenching agent, 2.5 g/L-40 g/L of aquenching agent, 2.75 g/L-40 g/L of a quenching agent, 3 g/L-40 g/L of aquenching agent, 3.25 g/L-40 g/L of a quenching agent, 3.5 g/L-40 g/L ofa quenching agent, 3.75 g/L-40 g/L of a quenching agent, 4 g/L-40 g/L ofa quenching agent, 4.25 g/L-40 g/L of a quenching agent, 4.5 g/L-40 g/Lof a quenching agent, 4.75 g/L-40 g/L of a quenching agent, 5 g/L-40 g/Lof a quenching agent, 5.25 g/L-40 g/L of a quenching agent, 5.5 g/L-40g/L of a quenching agent, 5.75 g/L-40 g/L of a quenching agent, 6 g/L-40g/L of a quenching agent, 6.25 g/L-40 g/L of a quenching agent, 6.5g/L-40 g/L of a quenching agent, 6.75 g/L-40 g/L of a quenching agent, 7g/L-40 g/L of a quenching agent, 7.25 g/L-40 g/L of a quenching agent,7.5 g/L-40 g/L of a quenching agent, 8 g/L-40 g/L of a quenching agent,8.25 g/L-40 g/L of a quenching agent, 8.5 g/L-40 g/L of a quenchingagent, 8.75 g/L-40 g/L of a quenching agent, 9 g/L-40 g/L of a quenchingagent, 9.25 g/L-40 g/L of a quenching agent, 9.5 g/L-40 g/L of aquenching agent, 9.75 g/L-40 g/L of a quenching agent, 10 g/L-40 g/L ofa quenching agent, 10.25 g/L-40 g/L of a quenching agent, 10.5 g/L-40g/L of a quenching agent, 10.75 g/L-40 g/L of a quenching agent, 11g/L-40 g/L of a quenching agent, 11.25 g/L

-   -   40 g/L of a quenching agent, 11.5 g/L-40 g/L of a quenching        agent, 11.75 g/L-40 g/L of a quenching agent, 12 g/L-40 g/L of a        quenching agent, 12.25 g/L-40 g/L of a quenching agent, 12.5        g/L-40 g/L of a quenching agent, 12.75 g/L-40 g/L of a quenching        agent, 13 g/L-40 g/L of a quenching agent, 13.25 g/L-40 g/L of a        quenching agent, 13.5 g/L-40 g/L of a quenching agent, 13.75        g/L-40 g/L of a quenching agent, 14 g/L-40 g/L of a quenching        agent, 14.25 g/L-40 g/L of a quenching agent, 14.5 g/L-40 g/L of        a quenching agent, 14.75 g/L-40 g/L of a quenching agent, or 15        g/L-40 g/L of a quenching agent.

The urine collection sample of the disclosure may also be pre-packedwith a chelating reagent. A chelator, or chelating reagent, is achemical that binds and holds on to (chelates) minerals and metals suchas chromium, iron, lead, mercury, copper, aluminum, nickel, zinc,calcium, cobalt, manganese, and magnesium. The urine collection tube ofthe disclosure can be packed with a chelator such asdiethylenetriaminepentaaceic acid (DTPA; C₁₄H₂₃O₁₀N₃),ethylenediaminetetraacetic acid (EDTA; C₁₀H₁₆O₈N₂),cyclohexaneediaminetetraacetic acid (CDTA; C₁₄H₂₂O₈N₂),ethylenediaminedi-o-hydroxuphenylacetic acid (EDDHA; C₁₈H₂₀O₈N₂),hydroxyethylethylenediaminetriacetic acid (HEDTA; C₁₀H₁₈O₇N₂),nitrilotriacetic acid (NTA; C₆H₉O₆N), ethylene glycol bis (2-aminoethylether) tetraacetic acid (EGTA; C₁₄H₂₄O₁₀N₂), citric acid (CIT; C₆H₂O₄),oxalic acid (OX; C₂H₂O₄), pyrophosphoric acid (P₂O₇; H₄P₂O₇), andtriphosphoric acid (P₃O₁₀; H₅P₃O₁₀). A urine collection tube of thedisclosure may be pre-packed with anywhere from: 1 mM to 500 mM of achelating agent, 1 mM to 490 mM of a chelating agent, 1 mM to 480 mM ofa chelating agent, 1 mM to 470 mM of a chelating agent, 1 mM to 460 mMof a chelating agent, 1 mM to 450 mM of a chelating agent, 1 mM to 440mM of a chelating agent, 1 mM to 430 mM of a chelating agent, 1 mM to420 mM of a chelating agent, 1 mM to 410 mM of a chelating agent, 1 mMto 400 mM of a chelating agent, 1 mM to 390 mM of a chelating agent, 1mM to 380 mM of a chelating agent, 1 mM to 370 mM of a chelating agent,1 mM to 360 mM of a chelating agent, 1 mM to 350 mM of a chelatingagent, 1 mM to 340 mM of a chelating agent, 1 mM to 330 mM of achelating agent, 1 mM to 320 mM of a chelating agent, 1 mM to 310 mM ofa chelating agent, 1 mM to 300 mM of a chelating agent, 1 mM to 290 mMof a chelating agent, 1 mM to 280 mM of a chelating agent, 1 mM to 270mM of a chelating agent, 1 mM to 260 mM of a chelating agent, 1 mM to250 mM of a chelating agent, 1 mM to 240 mM of a chelating agent, 1 mMto 230 mM of a chelating agent, 1 mM to 220 mM of a chelating agent, 1mM to 210 mM of a chelating agent, 1 mM to 200 mM of a chelating agent,1 mM to 190 mM of a chelating agent, 1 mM to 180 mM of a chelatingagent, 1 mM to 170 mM of a chelating agent, 1 mM to 160 mM of achelating agent, 1 mM to 150 mM of a chelating agent, 1 mM to 140 mM ofa chelating agent, 1 mM to 130 mM of a chelating agent, 1 mM to 120 mMof a chelating agent, 1 mM to 110 mM of a chelating agent, 1 mM to 100mM of a chelating agent, 1 mM to 90 mM of a chelating agent, 1 mM to 80mM of a chelating agent, 1 mM to 70 mM of a chelating agent, 1 mM to 60mM of a chelating agent, 1 mM to 50 mM of a chelating agent, 1 mM to 40mM of a chelating agent, 10 mM to 100 mM of a chelating agent, 20 mM to100 mM of a chelating agent, 30 mM to 100 mM of a chelating agent, 40 mMto 100 mM of a chelating agent, 50 mM to 100 mM of a chelating agent, 60mM to 100 mM of a chelating agent, 70 mM to 100 mM of a chelating agent,10 mM to 150 mM of a chelating agent, 20 mM to 150 mM of a chelatingagent, 30 mM to 150 mM of a chelating agent, 40 mM to 150 mM of achelating agent, 50 mM to 150 mM of a chelating agent, 60 mM to 150 mMof a chelating agent, or 70 mM to 150 mM of a chelating agent.

In preferred instances, each collection tube in the kit is pre-packedwith the formaldehyde, the quenching solution, the chelator, and asuitable amount of sodium azide to prevent bacterial growth in the tube.The volume of the pre-packaged solution can range from 0.5 millilitersto 4 milliliters depending on the size of the collection tube. Suchamounts provide for a concentrated amount of formaldehyde+quenchingsolution+chelator that is generally diluted about 5-fold by the additionof the urine sample when the subject attaches the urine collection tubeto the vacutainer. In many instances, an excess amount of aformaldehyde+quenching solution+chelator as provided in a vacutainer ofthe disclosure does not affect the ability of the particular biomarkerto be further analyzed, but a minimum amount of preservative isrequired. Pre-packaging in the vacutainers of the disclosure provides asuitable amount of a formaldehyde+quenching solution+chelator forcombining with a volume of urine that is in the range of 1 mL to 20 mLs;in the range of 1 mL to 15 mLs; in the range of 1 mL to 10 mLs; in therange of 1 mL to 5 mLs; or another suitable amounts within those ranges.

Further, in addition to cfDNA, the solution describes above alsostabilizes methylated cfDNA. DNA methylation is a common epigeneticmodification achieved by adding a methyl group to the fifth carbon ofcytosine (5-methylcytosine, 5 mC) via DNA methyltransferases (DNMTs).The current human genome build contains about 28 million CpGs, 60-80% ofwhich are methylated. Generally, the majority of all CpGs are methylatedin human, except short unmethylated regions called CpG islands (CGIs).Thus, methylated cfDNA can also be a biomarker for the presence of cfDNAin a sample.

In addition, methylation patterns of cfDNA can be consistent with theiroriginated cells or tissues. Since circulating nucleic acids canoriginate from different tissues, including an allograft, uniquemethylation patterns can be used to distinguish cfDNA originated fromdonor as compared to recipient. For instance, bisulfite sequencing (alsoknown as bisulphite sequencing) is the use of bisulfite treatment of DNAbefore routine sequencing to determine the pattern of methylation.Treatment of DNA with bisulfite converts cytosine residues to uracil,but leaves 5-methylcytosine residues unaffected. Therefore, DNA that hasbeen treated with bisulfite retains only methylated cytosines. Thus,bisulfite treatment introduces specific changes in the DNA sequence thatdepend on the methylation status of individual cytosine residues,yielding single-nucleotide resolution information about the methylationstatus of a segment of DNA. Various analyses can be performed on thealtered sequence to retrieve this information. Thus, such an analysiscan differentiate between single nucleotide polymorphisms from allograftcfDNA (cytosines and thymidine) as compared to single nucleotidespolymorphisms from the recipient of the transplant.

The disclosure provides kits and methods that effectively collect andpreserve cfDNA, including methylated forms of cfDNA, and protein for ananalysis that may occur several days after sample collection. Thus, thekits of the disclosure have included various pre-analytical factors suchas the type of urine collection tubes, ease of collection by anunassisted subject (e.g., subject at home without certified healthcareassistance), sample storage conditions (temperatures for home storageand shipping of samples via a courier service) and easy to followprotocol to increase compliance with a monitoring regimen. In somecases, the solutions describe herein provide a urine sample where thecfDNA and most proteins (total protein) are stable for at least 5 days,at least 6 days, at least 7 days, at least 8 days, at least 9 days, orat least 10 days at room temperature. Such stability provides anunprecedent ability for the subject to collect the urine sample athis/her own dwelling and ship it to a laboratory for subsequentanalysis.

Stabilizing Urine Biomarkers in Collection Tubes of the Disclosed Kits

C-X-C Motif Ligands, clusterin, and Creatine Collection Tubes

In some cases, a kit of the disclosure has a second urine collectiontube that is also pre-packaged with a stabilizing solution. In somecases, the cell free DNA may not be stable in the second urinecollection tube. Preferably, the second urine collection tube comprisesa chelator, a polyol, and sodium azide in a concentration sufficient toinhibit nucleases, cell lysis, and bacterial growth in the sample.

Chemokines and their corresponding receptors serve as inflammatory andmigratory signals for immune cells. Examples of these include CXCR3 andits corresponding ligands, CXCL9, CXCL10 and CXCL11, which participatein the induction of immune responses against several foreign antigens.Because renal allograft recipients are at continuous risk for numerousadverse conditions, including alloimmune rejection, detection of a riseinflammatory markers that threaten the long-term survival of theallograft can help identify organ rejection.

CXCL10 has been well established as a marker of immune-mediated injuryin a variety of contexts due to its role as a ligand for the CXCR3receptor. CXCL10 and cfDNA have also been shown to detect chronic lungallograft dysfunction in lung transplantation as well as rejection inkidney transplantation. Recent studies found that there is a significantnumber of patients with traditionally non-immune kidney diseases, suchas hypertension and type 2 diabetes, that had elevated CXCL10,potentially indicating a broader utility in the detection of early-stagekidney injury. Prior studies have identified type 2 diabetes to have asignificant CXCL10-mediated component and have identified endothelialcell-produced CXCL10 as a contributor to essential hypertension.

Clusterin, a glycoprotein with potent cohesive properties, is induced ina wide variety of acute and chronic experimental renal diseases.Clusterin mRNA is found in almost all mammal tissues and isconstitutively expressed in epithelial and neuronal cells, mainly at theinterface of fluid-tissue boundary of biologically active fluidsincluding digestive juice, semen, urine, cerebrospinal fluid (CSF), andplasma/serum. Clusterin mRNA and protein expression are regulated duringdevelopment and pathophysiologic processes and appear to be involved ina variety of stress responses as a biomarker of cellular senescence.Yet, on its own, clusterin has proved to be an insufficient andunreliable marker of renal damage, particularly allograft damage.

Creatinine is a waste product produced by muscles from the breakdown ofa compound called creatine. Creatinine is removed from the body by thekidneys. It is released at a constant rate by the body (depending onmuscle mass). Thus urinary creatinine is an index of muscle mass whenkidney function is normal. It is increased in body protein breakdown(catabolism), as in trauma and surgery. One gram of urinary creatinineis equivalent to about 17 to 20 kg body mass.

Symmetric dimethylarginine (SDMA) is a sensitive circulating kidneybiomarker whose concentrations in urine are believe to increase earlierthan creatinine as glomerular filtration rate decreases. Unlikecreatinine, SDMA is also believed to be unaffected by lean body mass. Ithas been studied in canine blood for early detection of decreasingkidney function in canines with chronic kidney disease (CKD).

ADMA is a metabolic by-product of continual protein modificationprocesses in the cytoplasm of all human cells. It is closely related toL-arginine, a conditionally essential amino acid. ADMA interferes withL-arginine in the production of nitric oxide (NO), a key chemicalinvolved in normal endothelial function and, by extension,cardiovascular health.

The second urine collection sample of the disclosure may be pre-packedwith a polyol, such as polyethylene glycol (PEG), glycerol, vaseline, oranother suitable polyol. The concentration of the polyol in the secondurine collection tube may be at least 40%, at least 50%, at least 60%,or at least 65% percent of the volume in the pre-packed second tube.

The second urine collection sample of the disclosure may be pre-packedwith a chelating reagent. The urine collection tube of the disclosurecan also be pre-packed with a chelator such asdiethylenetriaminepentaaceic acid (DTPA; C₁₄H₂₃O₁₀N₃),ethylenediaminetetraacetic acid (EDTA; C₁₀H₁₆O₈N₂),cyclohexaneediaminetetraacetic acid (CDTA; C₁₄H₂₂O₈N₂),ethylenediaminedi-o-hydroxuphenylacetic acid (EDDHA; C₁₈H₂₀O₈N₂),hydroxyethylethylenediaminetriacetic acid (HEDTA; C₁₀H₁₈O₇N₂),nitrilotriacetic acid (NTA; C₆H₉O₆N), ethylene glycol bis (2-aminoethylether) tetraacetic acid (EGTA; C₁₄H₂₄O₁₀N₂), citric acid (CIT; C₆H₂O₄),oxalic acid (OX; C₂H₂O₄), pyrophosphoric acid (P₂O₇; H₄P₂O₇), andtriphosphoric acid (P₃O₁₀; H₅P₃O₁₀). A urine collection tube of thedisclosure may be pre-packed with anywhere from: 1 mM to 100 mM of achelating agent, 1 mM to 90 mM of a chelating agent, 1 mM to 80 mM of achelating agent, 1 mM to 70 mM of a chelating agent, 1 mM to 60 mM of achelating agent, 1 mM to 50 mM of a chelating agent, 1 mM to 40 mM of achelating agent, 10 mM to 100 mM of a chelating agent, 20 mM to 100 mMof a chelating agent, 30 mM to 100 mM of a chelating agent, 40 mM to 100mM of a chelating agent, 50 mM to 100 mM of a chelating agent, 60 mM to100 mM of a chelating agent, 70 mM to 100 mM of a chelating agent, 10 mMto 150 mM of a chelating agent, 20 mM to 150 mM of a chelating agent, 30mM to 150 mM of a chelating agent, 40 mM to 150 mM of a chelating agent,50 mM to 150 mM of a chelating agent, 60 mM to 150 mM of a chelatingagent, or 70 mM to 150 mM of a chelating agent.

In preferred instances, each second collection tube in the kit ispre-packed with the polyol, the chelator, and a suitable amount ofsodium azide to prevent bacterial growth in the tube. The volume of thepre-packaged solution can range from 0.5 milliliters to 4 millilitersdepending on the size of the collection tube. Such amounts provide for aconcentrated amount of formaldehyde+quenching solution+chelator that isgenerally diluted about 5-fold by the addition of the urine sample whenthe subject attaches the urine collection tube to the vacutainer.

Using a Kit of the Disclosure to Monitor an Allograft

In most preferred embodiments, a kit and methods of the disclosure canbe used to collect and stabilize a combinations of biomarkers for highaccuracy monitoring of a solid organ of a subject, such as subject thatmay have received an allograft during an organ transplant, a subjectthat donated an allograft—and is otherwise healthy, or a subject thathas or is suspected of having a kidney injury, such as a kidney stone,CKD, a kidney injury caused by a virus (e.g., BK virus, Sars-CoV-2).

Following the initial technical challenge of implanting an organ in atransplantation procedure, maintaining the organ against a vast array ofpathologies for years to come, remains a challenge for all cliniciansworking in transplantation. Drug toxicity, opportunistic infection,primary disease recurrence, and the constant battle against organrejection are all differentials that are considered when graftdysfunction is observed, promoting a lifetime of laborious surveillance.

After organ transplantation, monitoring patients for evidence ofrejection is essential for mitigating graft loss. Diagnosis of rejectionof solid organ transplants traditionally requires needle biopsy andhistological assessment, which in some healthcare models can be costly,logistically challenging and carries the risk of procedure-relatedcomplications with associated morbidity. There remains a critical unmetneed for an easy to use, non-invasive product, that can provide morethan a mere inference of potential allograft injuries, but that is alsosensitive enough and accurate enough to eliminate the need for a needlebiopsy or histological assessment.

There are multiple challenges in requiring the recipient of an allograftto frequently be submitted to invasive procedures, many of which areexacerbated during pandemic and social restrictions. First, a sampleshould be obtained in a non-intrusive, or minimally intrusive manner.Second, the sample must be a source of informative biomarkers formonitoring transplant health and injuries. Third, there is a need fordetecting the biomarkers in a reliable, reproducible, and robust manner.Lastly, there is a need for an analysis of the data, which can requiretransforming data obtained by quantitative detection of biomarkers tocreate a composite score for a condition being studied, e.g. acuterejection (AR), allograft hypoxia, etc. The kits disclosed here overcomethe deficiencies of the current standard-of-care for transplantmonitoring, by stabilizing biomarkers for a sufficient period of timeafter sample collection.

Samples

The terms “biological sample” or “sample” as used herein, refers to amixture of cells, tissue, and liquids obtained or derived from anindividual that contains a cellular and/or other molecular entity thatis to be characterized and/or identified, for example based on physical,biochemical, chemical and/or physiological characteristics. In oneembodiment the sample is liquid (i.e., a biofluid), such as urine,blood, serum, plasma, saliva, phlegm, etc. In other embodiments, thesample is a histological section, such as a solid tissue section from abiopsy.

Machine Learning Processes

Machine-learning algorithms find and apply patterns in data.Multivariate machine learning, linear and nonlinear fitting algorithmshave been applied in biomarker searches. Machine learning is generallysupervised or unsupervised. In supervised learning, the most prevalent,the data is labeled to tell the machine exactly what patterns it shouldlook for. For instance, samples of a patient with a known diagnosis ofacute rejection are labeled as “acute rejection.” Samples from “normal”patients are labeled “normal.” The algorithm then starts looking forpatterns that are clearly distinct between “normal” and “acuterejection.”

In unsupervised learning, the data has no labels. The machine algorithmlooks for whatever patterns it can find. This can be interesting if, forinstance, every sample analyzed is from a subject who received anallograft. It could, for example, be used for detection of a broadallograft specific marker.

Subjects

A subject can be any human or animal, collectively “individuals”, suchas a subject that has received an allograft during an organ transplant.For instance, subjects can be humans, non-human primates such aschimpanzees, and other apes and monkey species; farm animals such ascattle, horses, sheep, goats, swine; domestic animals such as rabbits,dogs, and cats; laboratory animals including rodents, such as rats, miceand guinea pigs, and the like. A subject can be of any age. Subjects canbe, for example, elderly adults, adults, adolescents, pre-adolescents,children, toddlers, infants. In specific cases, a subject is a pediatricrecipient of an allograft.

A “subject”, also referred to as an “individual” can be a “patient.” A“patient,” refers to an subject who is under the care of a treatingphysician. In one embodiment, the patient is suffering from renal damageor renal injury. In another embodiment, the patient is suffering fromrenal disease or disorder. In another embodiment, the patient has had arenal transplant and is undergoing of renal graft rejection. In yetother embodiments, the patient has been diagnosed with renal injury,renal disease, or renal graft rejection, but has not had any treatmentto address the diagnosis.

Other Definitions

For purposes of interpreting this specification, the followingdefinitions will apply and whenever appropriate, terms used in thesingular will also include the plural and vice versa.

“Hybridization”, “probe hybridization”, “cfDNA probe hybridization” or“Alu probe hybridization” refers to a reaction in which one or morepolynucleotides react to form a complex that is stabilized via hydrogenbonding between the bases of the nucleotide residues. The hydrogenbonding may occur by Watson Crick base pairing, Hoogstein binding, or inany other sequence specific manner. The complex may comprise two strandsforming a duplex structure, three or more strands forming a multistranded complex, a single self-hybridizing strand, or any combinationof these. A hybridization reaction may constitute a step in a moreextensive process, such as the pairing with a cfDNA sequence (e.g.,probe hybridazation to an Alu region of a cfDNA), initiation of PCR, orthe cleavage of a polynucleotide by an enzyme. A sequence capable ofhybridizing with a given sequence is referred to as the “complement” ofthe given sequence.

As used herein, “stringent conditions” for hybridization refer toconditions under which a nucleic acid having complementarity to a targetsequence predominantly hybridizes with the target sequence, andsubstantially does not hybridize to non-target sequences. Stringentconditions are generally sequence-dependent, and vary depending on anumber of factors. In general, the longer the sequence, the higher thetemperature at which the sequence specifically hybridizes to its targetsequence. Non-limiting examples of stringent conditions are described indetail in Tijssen (1993). Laboratory Techniques In Biochemistry AndMolecular Biology-Hybridization With Nucleic Acid Probes Part I, SecondChapter “Overview of principles of hybridization and the strategy ofnucleic acid probe assay”, Elsevier, N.Y. Where reference is made to apolynucleotide sequence, then complementary or partially complementarysequences are also envisaged. These are preferably capable ofhybridising to the reference sequence under highly stringent conditions.Generally, in order to maximize the hybridization rate, relativelylow-stringency hybridization conditions are selected: about 20 to 25degrees Celsius. lower than the thermal melting point (T_(m)). The T_(m)is the temperature at which 50% of specific target sequence hybridizesto a perfectly complementary probe in solution at a defined ionicstrength and pH. Generally, in order to require at least about 85%nucleotide complementarity of hybridized sequences, highly stringentwashing conditions are selected to be about 5 to 15 degrees Celsiuslower than the T_(m). In order to require at least about 70% nucleotidecomplementarity of hybridized sequences, moderately-stringent washingconditions are selected to be about 15 to 30 degrees Celsius lower thanthe T_(m). Highly permissive (very low stringency) washing conditionsmay be as low as 50 degrees Celsius below the T_(m), allowing a highlevel of mis-matching between hybridized sequences. Those skilled in theart will recognize that other physical and chemical parameters in thehybridization and wash stages can also be altered to affect the outcomeof a detectable hybridization signal from a specific level of homologybetween target and probe sequences.

“Complementarity” refers to the ability of a nucleic acid to formhydrogen bond(s) with another nucleic acid sequence by eithertraditional Watson-Crick base pairing or other non-traditional types. Apercent complementarity indicates the percentage of residues in anucleic acid molecule which can form hydrogen bonds (e.g., Watson-Crickbase pairing) with a second nucleic acid sequence (e.g., 5, 6, 7, 8, 9,10 out of 10 being 50%, 60%, 70%, 80%, 90%, and 100% complementary).“Perfectly complementary” means that all the contiguous residues of anucleic acid sequence will hydrogen bond with the same number ofcontiguous residues in a second nucleic acid sequence. “Substantiallycomplementary” as used herein refers to a degree of complementarity thatis at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or100% over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 30, 35, 40, 45, 50, or more nucleotides, or refersto two nucleic acids that hybridize under stringent conditions.

The terms “polynucleotide”, “nucleotide”, “nucleotide sequence”,“nucleic acid” and “oligonucleotide” are used interchangeably. Theyrefer to a polymeric form of nucleotides of any length, eitherdeoxyribonucleotides or ribonucleotides, or analogs thereof.Polynucleotides may have any three dimensional structure, and mayperform any function, known or unknown. The following are non-limitingexamples of polynucleotides: coding or non-coding regions of a gene orgene fragment, loci (locus) defined from linkage analysis, exons,introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, shortinterfering RNA (siRNA), short-hairpin RNA (shRNA), micro-RNA (miRNA),ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides,plasmids, vectors, isolated DNA of any sequence, isolated RNA of anysequence, nucleic acid probes, and primers. The term also encompassesnucleic-acid-like structures with synthetic backbones, see, e.g.,Eckstein, 1991; Baserga et al., 1992; Milligan, 1993; WO 97/03211; WO96/39154; Mata, 1997; Strauss-Soukup, 1997; and Samstag, 1996. Apolynucleotide may comprise one or more modified nucleotides, such asmethylated nucleotides and nucleotide analogs. If present, modificationsto the nucleotide structure may be imparted before or after assembly ofthe polymer. The sequence of nucleotides may be interrupted bynon-nucleotide components. A polynucleotide may be further modifiedafter polymerization, such as by conjugation with a labeling component.

As used herein, the term “genomic locus” or “locus” (plural loci) is thespecific location of a gene or DNA sequence on a chromosome. A “gene”refers to stretches of DNA or RNA that encode a polypeptide or an RNAchain that has functional role to play in an organism and hence is themolecular unit of heredity in living organisms. For the purpose of thisinvention it may be considered that genes include regions which regulatethe production of the gene product, whether or not such regulatorysequences are adjacent to coding and/or transcribed sequences.Accordingly, a gene includes, but is not necessarily limited to,promoter sequences, terminators, translational regulatory sequences suchas ribosome binding sites and internal ribosome entry sites, enhancers,silencers, insulators, boundary elements, replication origins, matrixattachment sites and locus control regions.

As used herein, “expression of a genomic locus” or “gene expression” isthe process by which information from a gene is used in the synthesis ofa functional gene product. The products of gene expression are oftenproteins, but in non-protein coding genes such as rRNA genes or tRNAgenes, the product is functional RNA. The process of gene expression isused by all known life-eukaryotes (including multicellular organisms),prokaryotes (bacteria and archaea) and viruses to generate functionalproducts to survive. As used herein “expression” of a gene or nucleicacid encompasses not only cellular gene expression, but also thetranscription and translation of nucleic acid(s) in cloning systems andin any other context. As used herein, “expression” also refers to theprocess by which a polynucleotide is transcribed from a DNA template(such as into and mRNA or other RNA transcript) and/or the process bywhich a transcribed mRNA is subsequently translated into peptides,polypeptides, or proteins. Transcripts and encoded polypeptides may becollectively referred to as “gene product.” If the polynucleotide isderived from genomic DNA, expression may include splicing of the mRNA ina eukaryotic cell.

The terms “polypeptide”, “peptide” and “protein” are usedinterchangeably herein to refer to polymers of amino acids of anylength. The polymer may be linear or branched, it may comprise modifiedamino acids, and it may be interrupted by non amino acids. The termsalso encompass an amino acid polymer that has been modified; forexample, disulfide bond formation, glycosylation, lipidation,acetylation, phosphorylation, or any other manipulation, such asconjugation with a labeling component.

As used herein the term “amino acid” includes natural and/or unnaturalor synthetic amino acids, including glycine and both the D or L opticalisomers, and amino acid analogs and peptidomimetics.

As used herein the term “metabolite” refers to intermediate or endproducts of metabolism. The term metabolite is usually used for smallmolecules, but it can also include amino acids, vitamins, nucleotides,antioxidants, organic acids, and vitamins. In preferred cases, the termmetabolite refers to creatinine.

As used herein, the term “domain” or “protein domain” refers to a partof a protein sequence that may exist and function independently of therest of the protein chain.

As used herein, the terms “disorder” or “disease” and “injury” or“damage” are used interchangeably. It refers to any alteration in thestate of the body or one of its organs and/or tissues, interrupting ordisturbing the performance of organ function and/or tissue function(e.g., causes organ dysfunction) and/or causing a symptom such asdiscomfort, dysfunction, distress, or even death to a subject afflictedwith the disease.

A subject “at risk” of developing renal injury, renal disease or renalgraft rejection may or may not have detectable disease or symptoms, andmay or may not have displayed detectable disease or symptoms of diseaseprior to the treatment methods described herein. “At risk” denotes thata subject has one or more risk factors, which are measurable parametersthat correlate with development of renal injury, renal disease, or renalgraft rejection, as described herein and known in the art. A subjecthaving one or more of these risk factors has a higher probability ofdeveloping renal injury, renal disease, or renal graft rejection than asubject without one or more of these risk factor(s).

The term “diagnosis” is used herein to refer to the identification orclassification of a medical or pathological state, disease or condition.For example, “diagnosis” may refer to identification of renal injury,renal disease, or renal graft rejection. “Diagnosis” may also refer tothe classification of a severity of the renal injury, renal disease, orrenal graft rejection. Diagnosis of the renal injury, renal disease,chronic kidney disease (CKD), or renal graft rejection may be madeaccording to any protocol that one of skill of art (e.g., anephrologist) would use.

The term “companion diagnostic” is used herein to refer to methods thatassist in making a clinical determination regarding the presence, degreeor other nature, of a particular type of symptom or condition of renalinjury, renal disease, or renal graft rejection. For example, acompanion diagnostic of renal injury, renal disease, or renal graftrejection can include measuring the fragment size of cell free DNA.

The term “prognosis” is used herein to refer to the prediction of thelikelihood of the development and/or recurrence of an injury beingtreated with an allograft, e.g., a renal injury, renal disease, or renalgraft rejection. The predictive methods of the invention can be usedclinically to make treatment decisions by choosing the most appropriatetreatment modalities for any particular patient. The predictive methodsof the present invention are valuable tools in predicting if and/oraiding in the diagnosis as to whether a patient is likely to developrenal injury, renal disease, or renal graft rejection, have recurrenceof renal injury, renal disease, or renal graft rejection, and/orworsening of renal injury, renal disease, or renal graft rejectionsymptoms.

“Treating” and “treatment” refers to clinical intervention in an attemptto alter the natural course of the individual and can be performedbefore, during, or after the course of clinical diagnosis or prognosis.Desirable effects of treatment include preventing the occurrence orrecurrence of renal injury, renal disease, or renal graft rejection or acondition or symptom thereof, alleviating a condition or symptom ofrenal injury, renal disease, or renal graft rejection, diminishing anydirect or indirect pathological consequences of renal injury, renaldisease, or renal graft rejection, decreasing the rate of renal injury,renal disease, or renal graft rejection progression or severity, and/orameliorating or palliating the renal injury, renal disease, or renalgraft rejection. In some embodiments, methods and compositions of theinvention are used on patient sub-populations identified to be at riskof developing renal injury, renal disease, or renal graft rejection. Insome cases, the methods and compositions of the invention are useful inattempts to delay development of renal injury, renal disease, or renalgraft rejection. Beneficial or desired clinical results are known or canbe readily obtained by one skilled in the art. For example, beneficialor desired clinical results can include, but are not limited to, one ormore of the following: monitoring of renal injury, detection of renalinjury, identifying type of renal injury, helping renal transplantphysicians to decide whether or not to send transplant patients to gofor a biopsy and make decisions for the purposes of clinical managementand therapeutic intervention.

EXAMPLES Example 1: Stabilized Cell Free Nucleic Acids in Urine forShipping Via a Courier

Preservation of the integrity of cell free nucleic acids in urinespecimens during shipping and handling is crucial for remote monitoringof subjects. An economical and convenient method is described fornucleic acid stabilization by using a protein/nucleic acid stabilizingsolution in urine that is designed for the shipment of samples to alaboratory.

A subject receives a kit of the disclosure in the mail, via a courier.The kit comprises two 12 ml urine sample collection tubes pre-packagedwith the “first” pre-packaged solution and one 5 ml urine samplecollection tube pre-packaged with the “second” pre-packaged solution.the following instructions for use:

1. Prepping the Sample

A. Take all 12 components out of the box.

B. Clean genitals with alcohol wipe prior to collecting sample.

2. Collecting the Samples

A. Insert large tube, with white cap down, into urine collection cupopening and push all the way down for at least 5 seconds. The tube willfill on its own. Remove the tube and flip it back and fourthapproximately 10 times. Optionally, the insert instructions will state”

“Do not remove the white cap on the tube and do not consume the contentsof the tube.”

B. Insert 2nd large tube and repeat. Insert small tube and repeat.

C. Stick one label on each tube.

3. Packaging Samples

A. Insert the 3 tubes into the white sleeve; sleeve into the biohazardbag; and seal the bag.

B. Wrap the Therapak gel pack around the biohazard bag and place insidethe foil pouch.

C. Discard remaining urine in toilet and throw sample cup away.

4. Shipping Samples

Put the foil pouch into your kit box and put the kit box in the providedFedEx shipping bag. Place pre-made shipping label on the FedEx bag.

Optionally, a subject calls a Transplant Account Specialist to schedulea FedEx pick up

Example 2: Stability of Markers in Solution

Circulating cell-free DNA (cfDNA) in blood plasma derived from tumor,fetus and transplanted organs has been extensively studied. CirculatingcfDNA passes from blood, through the kidney barrier, and into urine.However, the inherent instability of cfDNA in urine has previouslyhindered its clinical utility as a biomarker. Nucleated cells in urinecould also release genomic DNA into urine leading to an increased highmolecular weight DNA background during sample processing and storage.NephroSant has a urine preservative for stabilizing cfDNA and the otherbiomarkers that comprise the QSant test in urine.

Preanalytical studies were performed to confirm effective preservativereagent(s) in urine post specimen collection can preserve cfDNA/m-cfDNA,Creatinine, Total Protein, Clusterin and CXCL10 up to 3 days (72 hours)at room temperature (RT). This is essential to ensure stability ofclinical specimens during shipment to NephroSant CLIA lab via FedExovernight priority shipment. The specimen shipper kit included a gelpack that maintains RT during the shipping process.

Briefly, at least one urine sample collection tube having a volume of apre-packaged solution for stabilizing a biomarker in the urine samplewas utilized to stabilize a biomarker in the sample. A first urinecollection tube had from 25 g/L-800 g/L of a formaldehyde donor, from2.5 g/L-80 g/L of a quenching agent, from 1 mM to 500 mM of a chelatingagent, and a suitable amount of sodium azide to prevent bacterial growth(optional sodium azide)(Preservative 1). A second urine collection tubewas pre-packed with at least 65% percent (volume) of a polyol, from 1 mMto 100 mM of a chelating agent, a crowding reagent such as Bovine SerumAlbumin (BSA), and a suitable amount of sodium azide to preventbacterial growth in the tube (optional sodium azide)(Preservative 3).

Results:

cfDNA and other QSant™ biomarker concentrations remained stable at RTfor at least 3 days in urine samples treated with urine preservative.

The disclosure validated urine preservatives that can effectivelystabilize urine biomarkers by conducting a time course. The studysubjected ˜30 patient samples (with high and low native levels)supplemented with a urine preservative and stored for 3 days at RTbefore testing.

Samples had high and low natural levels of cfDNA/m-cfDNA, Creatinine,Total Protein and Clusterin, but as endogenous levels of CXL10 were verylow, a proportion of the samples were spiked with high CXCL10 to cover ahigher range of concentration levels. FIGS. 10-14 illustrate theresults. Acceptable stability was observed up to 3 days of storage atroom temperature.

TABLE 1 QSant Biomarker Stability Study Summary TABLE 1 Average %Recovery with Preservative Biomarker (n samples) Day 3 cfDNA/m-cfDNA (n= 30) 102%  Clusterin (n = 24) 87% CXCL10 (n = 26) 97% Creatinine (n =29) 99% Total Protein (n = 28) 109% 

Percent recovery was calculated for each time-point according to theformula (% Recovery=Observed Concentration Day 3/Observed ConcentrationDay 0×100%).

Conclusion:

Our results show that addition of a urine preservative stabilizescfDNA/m-cfDNA, Clusterin, CXCL10, Total Protein and Creatinine in urinefor at least 3 days at room temperature. Thus, addition of thispreservative will allow for greater sample collection flexibility andreduce preanalytical variation during shipping and sample processing.

Example 3: Urine Preservatives

Circulating cell-free DNA (cfDNA) in blood plasma derived from tumor,fetus and transplanted organs has been extensively studied. CirculatingcfDNA passes from blood, through the kidney barrier, and into urine.However, the inherent instability of cfDNA in urine has previouslyhindered its clinical utility as a biomarker. Nucleated cells in urinecould also release genomic DNA into urine leading to an increased highmolecular weight DNA background during sample processing and storage.

The disclosure further optimized various alternatives of the urinepreservatives lacking the formaldehyde donor described in Example 2, forstabilizing cfDNA and the other biomarkers specifically in urinesamples. A second composition was validated that did not require aformaldehyde donor was validated for stabilizing cfDNA and methylatedcfDNA. The urine collection tube had from 2.5 g/L-80 g/L of a quenchingagent, from 1 mM to 500 mM of a chelating agent, and a suitable amountof sodium azide to prevent bacterial growth (optional) (Preservative 2).

Materials & Equipment

TABLE 2 Exemplary cfDNA reagents used for validation. TABLE 2Manufacturer Catalog ID Reagent Storage Requirement IDT Custom KIT ProbeALU (See, e.g., Sarwal −80° C. U. S. Pat. No. 11,124,824B2). PromegaCustom Sonicated Standard Control −80° C. Invitrogen AM9625 10X PBS RoomTemperature Thermo Fisher PI37525 Blocker BSA (10% in PBS) Refrigeration(2-8° C.) Scientific Thermo Fisher 37075 Super Signal ELISA FEMTORefrigeration (2-8° C.) Scientific Substrate R&D Systems DY998Streptavidin HRP Refrigeration (2-8° C.) Greiner 655074 LUMITRAC 600 HBmicroplate 96 Room Temperature well Thermo Fisher DY992 ELISA PlateSealers Room Temperature Scientific VistaLab Tech. 21381090 ReservoirRoom Temperature

TABLE 3 m-cfDNA reagents used for validation. TABLE 3 ManufacturerCatalog ID Reagent Storage Requirement Thermo Fisher MA5-246945-Methylcytosine Recombinant ≤−20° C. Scientific Rabbit MonoclonalAntibody (RM231) Thermo Fisher 32260 Goat anti-Rabbit Poly HRPRefrigeration (2-8° C.) Scientific Secondary Antibody In-house N/AsfmDNA Standard ≤−20° C. Invitrogen AM9625 10X PBS Room TemperatureThermo Fisher PI37525 Blocker BSA (10% in PBS) Refrigeration (2-8° C.)Scientific Thermo Fisher 37075 Super Signal ELISA Femto Refrigeration(2-8° C.) Scientific Substrate R&D Systems DY998 Streptavidin HRPRefrigeration (2-8° C.) Greiner 655074 LUMITRAC 600 HB microplate 96Room Temperature well Thermo Fisher DY992 ELISA Plate Sealers RoomTemperature Scientific VistaLab Tech. 21381090 Reservoir RoomTemperature

TABLE 4 Extraction reagents for QIASymphony. TABLE 4 ManufacturerCatalog ID Reagent Storage Requirement Qiagen 55114 QIAamp CirculatingNucleic Acid kit Room Temperature Qiagen **QIAGEN Mini Columns RoomTemperature Qiagen **Tube extenders (20 mL) Room Temperature Qiagen**Collection tubes (2.0 mL) Room Temperature Qiagen **Elution tubes (1.5mL) Room Temperature Qiagen **VacConnectors Room Temperature Qiagen**Buffer ACL Room Temperature Qiagen **Buffer ACB Room TemperatureQiagen **Buffer ACW1 Room Temperature Qiagen **Buffer ACW2 RoomTemperature Qiagen **Buffer AVE Room Temperature Qiagen **QIAGENProteinase K Refrigeration (2-8° C.) Qiagen **Carrier RNA RoomTemperature Qiagen 939016 Buffer ATL Room Temperature Fisher A4094Ethanol Room Temperature Chemical Fisher A416P4 Isopropanol RoomTemperature Chemical

TABLE 4 Extraction reagents for QIASymphony ™. TABLE 4 cfDNA Avg Conc.Sample Treatment Timepoint Avg RLU (GE/mL) % Recovery NUP383Preservative 1 Day 0 5,880,121 1,472.19 100% Day 1 6,886,189 1,729.98118% Day 4 6,503,755 1,631.98 111% Preservative 2 Day 0 4,346,5581,079.23 100% Day 1 6,646,464 1,668.55 155% Day 4 8,306,339 2,093.87194% NUP384 Control (No Preservative) Day 0 610,625 121.96 100% Day 3337,367 51.94  43% Preservative 1 Day 0 1,442,210 335.04 100% Day 328,098,385 7,165.29 2139%  Preservative 2 Day 0 5,736,603 1,435.41 100%Day 3 99,112 −9.11  −1% NUP385 Control (No Preservative) Day 037,820,221 9,656.37 100% Day 3 82,174 −13.45  0% Preservative 1 Day 0101,691 −8.45 100% Day 3 11,548,671 2,924.67 −34611%    Preservative 2Day 0 368,572 59.93 100% Day 3 13,553,054 3,438.26 5737%  NUP386 Control(No Preservative) Day 0 153,960 4.94 100% Day 3 883,897 191.98 3886% Preservative 1 Day 0 370,242 60.36 100% Day 3 4,906,278 1,222.65 2026% Preservative 2 Day 0 3,594,331 886.49 100% Day 3 5,539,602 1,384.93 156%NUP387 Control (No Preservative) Day 0 133,955 11.44 100% Day 3 99,4433.68  32% Preservative 1 Day 0 83,728 0.14 100% Day 3 439,915 80.2557321%  Preservative 2 Day 0 66,165 −3.81 100% Day 3 726,278 144.66−3797%  NUP388 Control (No Preservative) Day 0 50,290 −7.38 100% Day 3125,388 9.51 −129%  Preservative 1 Day 0 60,230 −5.14 100% Day 3 284,59145.32 −882%  Preservative 2 Day 0 91,016 1.78 100% Day 3 509,108 95.825383%  NUP389 Control (No Preservative) Day 0 3,048,123 666.86 100% Day3 4,430,467 977.77 147% Preservative 1 Day 0 10,042,712 2,240.01 100%Day 3 10,281,310 2,293.68 102% Preservative 2 Day 0 7,974,249 2,183.71100% Day 3 11,577,127 2,585.12 118%

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

What is claimed is:
 1. A kit for the stabilization of a urine sample from a subject comprising: a vacutainer cup for the collection of the urine sample from the subject, the vacutainer cup having an inner protrusion functionally connected to a piercing hollow channel; and two or more urine sample collection tubes having a volume of a pre-packaged solution, a pre-packaged powder, or a pre-packaged gel for stabilizing two or more distinct analytes in the urine sample, wherein a first analyte in the two or more distinct analytes is a cell-free nucleic acid, whereby the two or more urine sample collection tubes have a top configured to form a suction vacuum when pierced by the piercing hollow channel.
 2. The kit of claim 1, wherein a second analyte is a protein.
 3. The kit of claim 1, wherein the two or more distinct analytes are stable at temperatures up to 86° F. in their distinct two or more urine sample collection tubes.
 4. The kit of claim 1, wherein the pre-packaged solution, the pre-packaged powder, or the pre-packaged gel is for stabilizing a cell-free nucleic acid in the urine sample and a protein in the urine sample.
 5. The kit of claim 1, wherein the kit comprises a third urine sample collection tube.
 6. The kit of claim 5, wherein the third urine sample collection tube comprises a volume of a second pre-packaged solution, a pre-packaged powder, or a pre-packaged gel for stabilizing at least one additional biomarker.
 7. The kit of claim 1, wherein one of the two or more urine sample collection tubes is pre-packaged with a solution for stabilizing an inflammation marker in the urine sample.
 8. The kit of claim 7, wherein the inflammation marker is CXCL10 or CXCL9.
 9. The kit of claim 1, wherein one of the two or more urine sample collection tubes is pre-packaged with a solution is for stabilizing an apoptotic marker in the urine sample.
 10. The kit of claim 9, wherein the apoptotic marker in the urine sample is clusterin.
 11. The kit of claim 1, wherein one of the two or more urine sample collection tubes is pre-packaged with a solution for stabilizing a metabolite in the urine sample.
 12. The kit of claim 11, wherein the metabolite in the urine sample is creatinine.
 13. The kit of claim 1, wherein the kit further comprises an envelope, a box, or a bag for shipping the at least one urine sample collection tube after urine collection via a courier service.
 14. The kit of claim 13, wherein the envelope, the box, or the bag are pre-addressed for postage to a urine analysis laboratory via the courier.
 15. The kit of claim 1, wherein the postage to the urine analysis laboratory is pre-paid.
 16. The kit of claim 1, wherein the volume of the pre-packaged solution ranges from 0.5 milliliters to 4 milliliters.
 17. The kit of claim 1, wherein the volume of the pre-packaged solution provides for a 5 fold dilution of the urine sample.
 18. The kit of claim 1, wherein the urine sample collection tube is a 5 milliliter collection tube, a 10 milliliter collection tube, or a 12 milliliter collection tube.
 19. The kit of claim 1, wherein the subject has received an allograft.
 20. The kit of claim 19, wherein the allograft is kidney allograft. 